Multiplex amplification of polynucleotides

ABSTRACT

The present invention provides methods, reagents and kits for carrying out a variety of assays suitable for analyzing polynucleotides or samples that include an amplification step performed in a multiplex fashion. Also provided are methods for analyzing and improving the efficiency of amplification and for carrying out gene expression analysis.

1. CROSS-REFERENCE TO RELATED CO-PENDING APPLICATIONS

This application is a division of U.S. application Ser. No. 14/752,396,filed Jun. 26, 2015, which is a continuation of U.S. application Ser.No. 13/645,264, filed Oct. 4, 2012, now, abandoned, which is acontinuation of U.S. application Ser. No. 10/723,520, filed Nov. 26,2003, now U.S. Pat. No. 8,323,897, issued Dec. 4, 2012, which claims thebenefit of priority under 35 U.S.C. § 119(e) to application No.60/431,156, filed Dec. 4, 2002 and application No. 60/525,284, filedNov. 25, 2003, the disclosures of which are incorporated herein byreference.

2. FIELD OF THE INVENTION

The present invention relates to the field of molecular biology, and inparticular provides methods, reagents and kits for amplifyingpolynucleotide sequences of interest in a multiplex fashion. Onceamplified, the multiplex amplification product can be used in downstreamanalyses without further purification or manipulation.

3. BACKGROUND OF THE INVENTION

The general principles and conditions for amplification of nucleic acidsusing polymerase chain reaction are well known in the art (e.g., U.S.Pat. Nos. 4,683,195; 4,683,202; and 4,965,188). Amplification of nucleicacids from tissue samples represents an invaluable resource for bothdiagnosis and prognosis determinations, as well as the ability tocorrelate disease states with genetic disorders, including singlenucleotide polymorphisms (SNPs), aberrant gene expression, chromosomaland gene rearrangement, translocation and/or alternate splicing, andchromosomal duplication/elimination. However, conventional polymerasechain reaction methods allow for the amplification of only a single DNAtarget species per PCR reaction (e.g., singe-plex PCR). For example, theamplification of 10,000 target sequences of interest would typicallyrequire 10,000 separate polymerase chain reactions based on conventionalPCR procedures. The conventional approaches prove both time consumingand costly. There is, accordingly, a need in the art for improvedmethods of amplifying target sequences from a sample wherein anyplurality of target sequences may be amplified simultaneously underidentical reaction conditions in a multiplex fashion.

Moreover, certain downstream assays, such as array-based assays andquantitative PCR assays require a significantly high quantity ofstarting sample of target nucleic acid for carrying out the appropriateanalyses. In situations where only a limited quantity of starting sampleis available for use, only one or a few downstream analyses may beperformed before the sample is depleted. There is, accordingly, a needin the art for methods which amplify, or significantly increase, thequantity of the starting material for permitting a variety of downstreamassays to be carried out, optionally simultaneously, for providing avariety of information about a sample of interest in a relatively briefperiod of time.

4. SUMMARY OF INVENTION

In various embodiments, the present invention provides methods, reagentsand kits for amplifying polynucleotide sequences of interest in amultiplex fashion. According to one embodiment of the methods of theinvention, one or more polynucleotides are amplified, for example by thepolymerase chain reaction (“PCR”) or reverse-transcription polymerasechain reaction (“RT-PCR”), using a plurality of amplification primerpairs or sets, each of which is suitable or operative for amplifying adifferent polynucleotide sequence of interest. Unlike conventionalamplification reactions, which are carried out with a single pair or setof amplification primers, and therefore generate a single amplifiedsequence (“amplicon”), by virtue of utilizing a plurality ofamplification primer pairs or sets, the multiplex amplification methodsof the invention permit the simultaneous amplification of a plurality ofdifferent sequences of interest in a single reaction.

As will be discussed in more detail, below, because a plurality ofdifferent sequences are amplified simultaneously in a single reaction,the multiplex amplifications may be used in a variety of contexts toeffectively increase the concentration or quantity of a sample availablefor downstream analyses and/or assays. Once the sample has beenmultiplex amplified according to the methods described herein, it may bedivided into aliquots, with or without prior dilution, for subsequentanalyses. Owing to its increased concentration and quantity,significantly more analyses or assays can be performed with themultiplex amplified sample than could have been performed with theoriginal sample. In many embodiments, multiplex amplification evenpermits the ability to perform assays or analyses that require moresample, or a higher concentration of sample, than was originallyavailable. For example, after a 1000× multiplex amplification,subsequent assays could then be performed at 1000× less sample volume.Concentrating by multiplex amplification can also be used to dilute outamplification inhibitors that may be present in the original sample.

Although the multiplex amplification reactions may be carried out usingconventional PCR reagents, reaction conditions and cycle temperaturesand times, it has been discovered that the amounts of the variousamplicons generated in the multiplex amplification reaction can beincreased by increasing the amount or concentration of DNA polymeraseused and/or the length of the time or duration of the primer extensionreaction per cycle.

In certain embodiments, in generating the multiplex amplificationproducts, the relative concentrations of the various amplicons generatedduring the amplification can be maintained sufficiently for detectingchanges in the relative concentrations by increasing the amount orconcentration of DNA polymerase used and/or the length of the time orduration of the primer extension reaction per cycle.

Moreover, while conventional primer concentrations can be used, it hasbeen surprisingly discovered that the amplification proceeds with a highdegree of efficiency in the presence of very low concentrations ofamplification primers. By way of comparison, whereas conventionalsingle-plex (“simplex”) PCR amplifications are carried out in thepresence of 300-900 nM each primer, highly efficient multiplexamplification was achieved with only 45 nM of each primer.

Both DNA and RNA target polynucleotides can be multiplex amplified usingsuch low primer concentrations. Specifically, the reverse transcriptionof RNA into cDNA via a reverse-transcription and subsequent multiplexamplification of the resultant cDNA with a DNA polymerase may beaccomplished using primers at low concentrations (e.g., 45 nM for eachprimer). Accordingly, the invention permits the amplification of bothDNA and RNA target polynucleotides in a multiplex fashion usingprinciples of conventional polymerase chain reactions (PCR) andreverse-transcription polymerase chain reactions (RT-PCR), respectively.

In addition, the individual primer concentrations do not need to beoptimized; it has been discovered that using all primers atapproximately equimolar concentrations yields good results. It was,moreover, demonstrated in particular embodiments of the invention thatthe use of low primer concentrations reduces the possibility ofnon-specific primer interactions (e.g., primer dimerization), therebyeliminating the need for optimization. The concentrations of primersdescribed in various embodiments of the invention (e.g., 45 nM, eachprimer) were demonstrated to be sufficiently high to permit themultiplex amplification of target sequences yet low enough to prevent oravoid the primers from interacting non-specifically with one another.Thus, multiplex amplification of virtually any combination of sequencescan be rapidly achieved without time-consuming optimization steps.

It has also been discovered that the presence of oligonucleotide probesin the multiplex amplification does not significantly interfere with theamplification reactions. Thus, the multiplex amplification can beeffectively carried out in the presence of oligonucleotide probes, suchas, for example, non-priming oligonucleotide probes designed forquantitative or real-time PCR analysis. Non-limiting examples of typesof probes that can be present in the multiplex amplification includeTaqMan® probes (see, e.g., U.S. Pat. No. 5,538,848), stem-loop orhairpin Molecular Beacons™ (see, e.g., U.S. Pat. Nos. 6,103,476 and5,925,517 and Tyagi & Kramer, 1996, Nature Biotechnology 14:303-308),stemless or linear beacons (see, e.g., WO 99/21881), PNA MolecularBeacons (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNAbeacons (see, e.g., Kubista et al., 2001, SPIE 4264:53-58), non-FRETprobes (see, e.g., U.S. Pat. No. 6,150,097), Sunrise®/Amplifluor® probes(U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion™ probes(Solinas et al., 2001, Nucleic Acids res. 29:E96 and U.S. Pat. No.6,589,743), bulge loop probes (U.S. Pat. No. 6,590,091), pseudo knotprobes (U.S. Pat. No. 6,548,250), cyclicons (U.S. Pat. No. 6,383,752),MGB Eclipse™ probe (Epoch Biosciences), hairpin probes (U.S. Pat. No.6,596,490), peptide nucleic acid (PNA) light-up probes, self-assemblednanoparticle probes, and ferrocene-modified probes.

This discovery is significant, as it permits the ability to carry outmultiplex amplification reactions using off-the-shelf commerciallyavailable reagents, such as the gene expression and SNP reagents soldunder the tradename Assays-On-Demand® by Applied Biosystems (an AppleraCorporation business; Foster City, Calif.). In this context,off-the-shelf reagents comprising amplification primers and probes canbe pooled together and used in a multiplex amplification reactionwithout prior removal of probes. Like multiplex amplifications carriedout in the absence of such oligonucleotide probes, multiplexamplifications carried out in the presence of such oligonucleotideprobes can be divided into aliquots, with or without prior dilution, forsubsequent analysis without further purification or manipulation.

Samples amplified in a multiplex fashion may be used in virtually anysubsequent analysis or assay without further purification ormanipulation. For example, the product of the multiplex amplificationmay be used for single polynucleotide polymorphism (“SNP”) analysis,genotyping analysis, gene expression analysis, fingerprinting analysis,analysis of gene mutations for genetic diagnoses, analysis of rareexpressed genes in cells, nucleic acid sequencing (e.g., U.S. Pat. No.6,428,986), nucleic acid mini-sequencing (e.g., U.S. Pat. No.6,479,242), and for hybridizing to arrays (e.g., U.S. Pat. No.6,485,944). The multiplex amplification product is even suitable forfurther amplification analyses, such as analysis via quantitative orreal-time PCR amplification. In this latter embodiment, the sequences ofthe primers used for the subsequent quantitative or real-time PCRanalyses can be the same as or different from those employed in theinitial multiplex amplification. The multiplex amplification product canbe divided into aliquots each of which can be subject to subsequentsingle-plex or multiplex assays or analyses. Alternatively, themultiplex amplification product need not divided into aliquots, andvarious assays and analyses can be performed directly on the product.

Significantly, such subsequent analyses or assays can be carried outdirectly with the multiplex amplification product without having tofirst remove the pooled oligonucleotide probes and/or primers. Anyprobes and/or primers carried over into the subsequent analysis orassays will not interfere with the analysis or assay.

In yet another embodiment, the present invention provides a two-stepmethod of analyzing a sample for the presence of one or morepolynucleotide sequences of interest. In a first step, one or morepolynucleotides derived from the sample (or a plurality of differentsamples) are multiplex amplified in the presence of a plurality ofdifferent amplification primer pairs or sets, as described above. In oneembodiment, in a second step, the product of the multiplex amplificationis single-plex amplified by polymerase chain reaction in the presence ofa set of amplification primers operative or suitable for amplifying asequence of interest and the single-plex amplification reactionmonitored for accumulation of amplification product. In anotherembodiment, in a second step the multiplex amplification product isdivided into a plurality of reaction vessels, the product in each vesselis single-plex amplified in the presence of a set of amplificationprimers operative or suitable for amplifying a sequence of interest andthe single-plex amplifications monitored for the accumulation ofamplification product. In either embodiment, the accumulation ofsingle-plex amplification product indicates the sample contains therespective polynucleotide of interest.

The accumulation of single-plex amplification product can be monitoredat the end of the reaction by conventional means, e.g., bychromatography, by eletrophoresis, by staining or by the use of asequence specific hybridization probe (e.g., a fluorescently labeledprobe). Alternatively, the accumulation of single-plex amplificationproduct can be monitored as a function of time using well known methods,such as carrying out the single-plex amplification in the presence ofone or more dyes or labels capable of producing a detectable signal uponbinding double-stranded polynucleotide (e.g., SYBR® Green I or II, SYBR®Gold, ethidium bromide, or YO-PRO-1; Molecular Probes, Eugene, Oreg.) oran oligonucleotide probe labeled with a suitable labeling system (e.g. aTaqMan® probe, or one of the various different types of exemplary probesdescribed above).

The present invention also provides reagents and kits suitable forcarrying out the multiplex amplifications and optional downstreamanalyses. In one embodiment, the kit includes a plurality ofamplification primer sets suitable for carrying out a multiplexamplification packaged in a single container. The kit may optionallyinclude one or more additional reagents for carrying out theamplification, such as a DNA polymerase enzyme, a reverse transcriptaseenzyme and/or mixtures of nucleoside triphosphates (“dNTPs”) suitablefor extension of the primers via template-dependent DNA synthesis. Theamount of optional polymerase included in the kit may be suitable foroptimizing the efficiency of the multiplex amplification reaction. Thevarious reagents may be packaged in combinations for maximalconvenience, and may be modeled after the combinations of reagentsavailable commercially for carrying out conventional PCR and/or RT-PCRamplification reactions (e.g., (2×) TaqMan® Universal PCR Master Mix andTaqMan® Gold RT-PCR Kit available from Applied Biosystems, an AppleraCorporation business). The kit may further include reagents useful forcarrying out downstream assays or analyses with the multiplexamplification product. For example, the kit may further includeoligonucleotide probes useful for SNP detection or analysis,oligonucleotide microarrays, such as microarrays suitable for geneexpression or SNP analyses, and/or “tailed” primers (see, e.g., Bengraet al., 2002, Clin. Chem. 48:2131-2140; Myakishev et al., 2001, GenomeRes. 11:163-169; and U.S. Pat. No. 6,395,486) for universalamplification, detection and/or purification. In one embodiment, the kitfurther includes reagents suitable for carrying out a plurality ofsingle-plex quantitative or real-time amplification reactions. Suchreagents typically include a set of quantitative or real-timeamplification primers, an oligonucleotide probe labeled with a labelingsystem suitable for monitoring the quantitative real-time amplificationreaction, a DNA polymerase at a concentration suitable for single-plexamplification and/or mixtures of dNTPs suitable for template-dependentDNA synthesis. The kit may include one or more of any of theseadditional reagents.

Various embodiments of the multiplex amplification methods, reagents andkits of the present invention provide significant advantages to thestate-of-the-art. For example, by virtue of the use of a plurality ofamplification primers, some embodiments of the multiplex amplificationspermit amplification of polynucleotide samples of limited quantity orcopy number, thereby permitting, for the first time, the ability toperform one or more downstream analyses that would otherwise, owing tolimitations of the sample quantity, be extremely difficult, timeconsuming, inaccurate or even unattainable. In certain embodiments,multiplex amplification can also permit target polynucleotide samples tobe concentrated, even in instances where the original sample included adilute pool of a plurality of target polynucleotides. Concentratingsamples in this way enables the ability to perform downstream analysesand assays that require highly concentrated samples, even in instanceswhere the original sample was too dilute. In further embodiments,multiplex amplifications can be performed in a single tube usingoff-the-shelf prepackaged reagents and permit the amplification ofvirtually any type of target polynucleotide sequence from virtually anytype of sample, wherein reagents and reaction conditions need not beoptimized to accommodate the amplification of a particular targetsequence within a particular sample. In many embodiments, the multiplexamplifications described herein were found to proceed with a high degreeof efficiency. The multiplex amplified product can therefore be used fordownstream analyses where the relative or absolute quantities of copynumbers in the starting sample are assessed, such as, for example,expression profiling analyses. Other advantages of various embodimentsof the present invention will be apparent upon review of the instantdisclosure.

5. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a cartoon illustrating the general principles of anembodiment of a multiplex amplification according to the invention;

FIG. 2 provides a cartoon illustrating an embodiment of a two-step assayaccording to the invention in which a multiplex amplification is coupledwith a plurality of downstream, single-plex (“simplex”) quantitative orreal-time PCR analyses;

FIG. 3 provides a graph illustrating the amplification efficiency of anembodiment of a 95-plex amplification reaction of the invention as afunction of DNA polymerase concentration;

FIG. 4 provides a graph illustrating the observed amplificationefficiency of an embodiment of a 95-plex amplification reaction of theinvention carried out with a total of 100 ng cDNA (from a cDNA library)and 6 U/20 μL AmpliTaq Gold® for a total of 10 cycles (cycle time wasapprox. 1 min./cycle);

FIG. 5 provides a graph demonstrating that the presence of a pluralityof amplification primers and oligonucleotide probes during an exemplarydownstream single-plex assay does not detrimentally affect theperformance of the assay;

FIG. 6 provides a graph demonstrating the linear relationship betweenamount of sample polynucleotide in a multiplex amplification and Ctvalue obtained during downstream individual real-time PCRamplifications;

FIG. 7 provides a bar graph illustrating the observed amplificationefficiency of downstream single-plex assay amplifications after variousmultiplex amplifications;

FIG. 8 provides a bar graph illustrating the effect of UNG in amultiplex amplification on observed amplification efficiency ofdownstream single-plex assay amplifications; and

FIG. 9 provides a graph illustrating the effect of increasing the numberof PCR cycles in multiplex amplifications on the Ct values of downstreamsingle-plex assay amplifications.

6. DETAILED DESCRIPTION

In certain embodiments, the present invention provides methods, reagentsand kits for amplifying polynucleotide sequences of interest in amultiplex fashion. The multiplex amplifications utilize well-knownprinciples and reagents for the amplification of DNA or RNApolynucleotides via the polymerase chain reaction (“PCR”) or thereverse-transcription polymerase chain reaction (“RT-PCR”),respectively, with one important difference. Rather than using a singleset or pair of amplification primers, the multiplex amplificationsutilize a plurality of different amplification primer pairs or sets in asingle reaction, permitting the simultaneous amplification of aplurality of polynucleotide sequences in a single reaction. Thus, ratherthan generating a single amplification product or “amplicon,” themultiplex amplifications generate a plurality of different amplicons ina single reaction. As will be described in more detail below,polynucleotides that can be amplified in a multiplex fashion includeboth 2′-deoxribonucleic acids (DNA) and ribonucleic acids (RNA). Whenthe polynucleotide to be amplified (“target polynucleotide”) is an RNA,it may be first reversed-transcribed to yield a cDNA, which can then beamplified in a multiplex fashion. Alternatively, the target RNA may bemultiplex amplified directly in the presence of the plurality ofamplification primer pairs using principles of RT-PCR. Accordingly, asused in the context of multiplex amplifications, “polymerase chainreaction” is meant to include both multiplex PCR and multiplex RT-PCR.

The principles of DNA amplification by PCR and RNA amplification byRT-PCR are well-known and described in myriad references, including:U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; 5,338,671;5,340,728; 5,405,774; 5,436,149; 5,512,462; 5,618,703; 6,037,129;6,300,073; and 6,406,891; Innis et al., 1990, In: PCR Protocols A guideto Methods and Applications, Academic Press, San Diego; and Schlesser etal., 1991, Applied and Environ. Microbiol. 57:553-556, the disclosuresof which are incorporated herein by reference. For ease of understandingthe differences and advantages of the multiplex amplifications describedherein, a brief summary of these amplification methods is provided.Conventional PCR requires at least two primers, a forward primer and areverse primer, which hybridize to a double-stranded targetpolynucleotide sequence to be amplified. In PCR, a double-strandedtarget DNA polynucleotide which includes the sequence to be amplified isincubated in the presence of the two amplification primers, a DNApolymerase and a mixture of 2′-deoxyribonucleotide triphosphates(“dNTPs”) suitable for DNA synthesis. To begin the amplification, thedouble-stranded target DNA polynucleotide is denatured and one primer isannealed to each strand of the denatured target. The primers anneal tothe target DNA polynucleotide at sites removed from one another and inorientations such that the extension product of one primer, whenseparated from its complement, can hybridize to the other primer. Once agiven primer hybridizes to its respective target DNA polynucleotidestrand, the primer is extended by the action of the DNA polymerase. Theextension product is then denatured from the target sequence, and theprocess is repeated. A PCR cycle (i.e. a thermocycle) typically includesthe steps of denaturation, annealing and extension.

In successive cycles of this process, the extension products produced inearlier cycles serve as templates for subsequent DNA synthesis.Beginning in the second cycle, the product of the amplification beginsto accumulate at a logarithmic rate. The final amplification product, or“amplicon,” is a discrete double-stranded DNA molecule consisting of:(i) a first strand which includes the sequence of the first primer,which is followed by the sequence of interest, which is followed by asequence complementary to that of the second primer, and (ii) a secondstrand which is complementary to the first strand.

Conventional RT-PCR permits the amplification of a target RNApolynucleotide sequence. In RT-PCR, a single-stranded RNA target whichincludes the sequence to be amplified (e.g., an mRNA) is incubated inthe presence of a reverse transcriptase, two amplification primers, aDNA polymerase and a mixture of dNTPs suitable for DNA synthesis. One ofthe amplification primers anneals to the RNA target and is extended bythe action of the reverse transcriptase, yielding an RNA/cDNAdoubled-stranded hybrid. This hybrid is then denatured, and the otherprimer annealed to the denatured cDNA strand. Once hybridized, theprimer is extended by the action of the DNA polymerase, yielding adouble-stranded cDNA, which then serves as the double-stranded templateor target for further amplification via conventional PCR, as describedabove. Thus, the main difference between conventional PCR and RT-PCR isthe presence of the reverse-transcriptase in the reaction mixture of thelatter. Following reverse transcription, the RNA can remain in thereaction mixture during subsequent PCR amplification, or it can beoptionally degraded by well-known methods prior to subsequent PCRamplification.

A PCR and/or RT-PCR amplification can incorporate one or more of avariety of improvements as described in the art. Non-limiting example ofsuch improvements include “hot start” PCR technique (D'Aquila et al.,1991, Nucl. Acids Res. 19:3749), “touchdown” PCR techniques (Don et al.,1991, Nucl. Acids Res. 19:4008), the use of polymerase enhancing factors(e.g. ArchaeMaxx™ available from Stratagene; see also U.S. Pat. No.6,444,428) and the use of dUTP in place of dTTP with treatment by uracilN-glycosylase (UNG) (as described in Example 6 herein, and see U.S. Pat.No. 5,035,996). Any of these improvements and/or other improvements forcarrying out PCR and/or RT-PCR amplifications can be used in conjunctionwith the various methods described herein.

A general overview of certain embodiments of the invention is providedin FIG. 1. Referring to FIG. 1, one or more target polynucleotides froma sample, which may be one or more RNAs or DNAs, is amplified by PCR orRT-PCR in the presence of a plurality of amplification primer pairs,each of which is suitable for amplifying a different target sequence ofinterest. In some embodiments, the number of primer pairs can be atleast 100, 300, 500, 1000, 10000, or 30000. As illustrated, when thesample or target polynucleotide is an RNA, the first cDNA strand may beoptionally synthesized prior to multiplex amplification using eitherrandom RT-primers (e.g., random hexamers or oligo (dT) primers) orsequence-specific RT primers, as is well known in the art.Alternatively, the target RNA may be directly multiplex amplified byRT-PCR in the presence of the plurality of amplification primer pairs.Owing to the presence of the plurality of amplification primer pairs,the product of the multiplex amplification is a plurality of differentamplicons, represented by amplicons 1 and 3.

As will be appreciated by skilled artisans, target polynucleotidessuitable for multiplex amplification may be either DNA (e.g., cDNA orgenomic DNA) or RNA (e.g., mRNA or rRNA) in nature, and may be derivedor obtained from virtually any sample or source, wherein the sample may,optionally, be scarce or of a limited quantity. For example, the samplemay be one or a few cells collected from a crime scene or a small amountof tissue collected via biopsy.

By way of example and not limitation, the target polynucleotide may be achromosome or a gene or a portion or fragment thereof, a regulatorypolynucleotide, a restriction fragment from, for example a plasmid orchromosomal DNA, genomic DNA, mitochondrial DNA, DNA from a construct orlibrary of constructs (e.g., from a YAC, BAC or PAC library), RNA (e.g.,mRNA, rRNA) or a cDNA or cDNA library. The target polynucleotide mayinclude a single polynucleotide, from which a plurality of differentsequences of interest may be amplified, or it may include a plurality ofdifferent polynucleotides, from which one or more different sequences ofinterest may be amplified. As will be recognized by skilled artisans,the sample or target polynucleotide may also include one or morepolynucleotides that are not amplified in the multiplex amplificationreaction.

An important embodiment of a multiplex amplification as described hereinis its ability to amplify polynucleotide sequences from highly complexmixtures of sample polynucleotides. Indeed, many embodiments aresuitable for multiplex amplification of target polynucleotide samplescomprising tens, hundreds, thousands, hundreds of thousands or evenmillions of polynucleotide molecules. In specific embodiments, themultiplex amplification methods can be used to amplify pluralities ofsequences from samples comprising cDNA libraries or total mRNA isolatedor derived from biological samples, such as tissues and/or cells,wherein the cDNA, or alternatively mRNA, libraries may be quite large.For example, cDNA libraries or mRNA libraries constructed from severalorganisms, or from several different types of tissues or organs, can bemultiplex amplified according to the methods described herein. As aspecific example, multiplex amplification from an extremely complexlibrary of cDNAs constructed from several different tissues or organswas achieved with good results (see, e.g., Example 4). It is believedthat this particular cDNA library included in the range of 10,000-20,000cDNAs.

The quantity of target polynucleotide amplified can vary widely. In manyembodiments quantities suitable for conventional PCR and/or RT-PCR maybe used. For example, the target polynucleotide may be from a singlecell, from tens of cells, from hundreds of cells or even more, as iswell known in the art. For many embodiments, including embodiments inwhich the target polynucleotide is a complex cDNA library, the totaltarget polynucleotide may range from about 1 pg to 100 ng.

Skilled artisans will appreciate that several advantages flow frommultiplex amplifications generally, and in particular multiplexamplifications carried out with complex target polynucleotides such asmRNA and/or cDNA libraries. First, samples including targetpolynucleotides that are present in low copy numbers can be effectivelyincreased in quantity, permitting the ability to carry out significantlymore downstream analyses or assays that would have been possible withoutmultiplex amplification. Second, the multiplex amplification permits theability to perform downstream analyses or assays that may not have beenpossible with the original sample owing to its limited quantity. Third,target polynucleotides within a large dilute pool or sample can beconcentrated, permitting the ability to perform downstream analyses orassays requiring highly concentrated samples. Moreover, as will bedescribed in more detail below in relation to certain embodiments, sincethe multiplex amplification proceeds with a high degree of efficiency,the relative concentrations of the various target sequences in thesample are sufficiently preserved to permit the ability to assess orquantify the relative concentrations in downstream analyses (e.g. indownstream gene expression profiling analyses).

The target polynucleotide(s) to be amplified can be prepared formultiplex amplification using conventional sample preparation techniquessuitable for the type of amplification reaction to be used. For example,the polynucleotides may be isolated from their source viachromatography, precipitation, electrophoresis, as is well-known in theart. Alternatively, the polynucleotide(s) may be amplified directly fromcells or from lysates of tissues or cells comprising the targetpolynucleotides. In some embodiments, the polynucleotide(s) to beamplified may be subjected to conventional sodium bisulphite treatment,or equivalent, in order to detect methylated cytosine residues (see,e.g., U.S. Pat. No. 6,596,488).

The number of sequences that may be amplified, or, stated another way,the number of amplicons that may be generated, by a multiplexamplification is dictated in large part by the number of differentamplification primer pairs used during the multiplex amplification.According to certain embodiments of the invention, each amplificationprimer pair includes two amplification primers, one forwardamplification primer and one reverse amplification primer, as iswell-known in the art. The amplification primer pairs may besequence-specific and may be designed to hybridize to sequences thatflank a sequence of interest to be amplified. Thus, the actualnucleotide sequences of each primer pair may depend upon the sequence ofinterest to be amplified, and will be apparent to those of skill in theart. Methods for designing primer pairs suitable for amplifying specificsequences of interest via PCR or RT-PCR are well-known. See e.g., Eckertet al. (1991) PCR: A Practical Approach, McPherson, Quirke, and Tayloreds., IRL Press, Oxford, Vol. 1, pp. 225-244; TaqMan® Universal PCRMaster Mix Protocol (available from Applied Biosystems, an AppleraCorporation business, Cat. #4304449 Rev. C); Rozen et al., 2000,Bioinformatics Methods and Protocols: Methods in Molecular Biology,Humana Press, Totowa, N.J., pp 365-386;http://www.ucl.ac.uk/wibr/2/services/reldocs/taqmanpr.pdf;http://www.ukl.uni-freiburg.de/core-facility/taqman/taqindex.html;http://www.operon.com/oligos/toolkit.php;http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi;http://www.ncbi.nlm.nih.gov/BLAST/; andhttp://www.biotech.uiuc.edu/primer.htm, which provide examplesdemonstrating how particular primer pairs may be designed.

Generally, each amplification primer must be sufficiently long to primethe template-directed synthesis of the target sequence under theconditions of the amplification reaction. The exact lengths of theprimers may depend on many factors, including but not limited to, thedesired hybridization temperature between the primers and templatepolynucleotides and the complexity of the different targetpolynucleotide sequences to be amplified. The ability to select lengthsand sequences of primers suitable for particular applications is withinthe capabilities of ordinarily skilled artisans. In certain embodiments,the primers may contain from about 15 to about 35 nucleotides, althoughthe primers may contain more or fewer nucleotides. Short primermolecules generally require lower temperatures to form sufficientlystable hybrid complexes with the template. Generally, the amplificationprimers should be designed to have a melting temperature (T_(m)) in therange of about 55-75° C. Melting temperatures in this range will tend toinsure that the primers remain annealed or hybridized to the targetpolynucleotide at the initiation of primer extension. The actualtemperature used for the primer extension reaction may depend upon,among other factors, the concentration of the primers which are used inthe multiplex assays. For amplifications carried out with a thermostablepolymerase such as Taq DNA polymerase, the amplification primers can bedesigned to have a T_(m) in the range of about 60 to about 78° C. Themelting temperatures of the different amplification primers can bedifferent; however, preferably they should all be approximately thesame.

T_(m) can be determined empirically using several standard procedures,based on ultraviolet hypochromism, for example, by monitoring thespectrum at 260 nm (e.g. as described in Biochemistry—The MolecularBasis of Cell Structure and Function, 2nd Edition, Lehninger, WorthPublishers, Inc., 1970, pp. 876-7). The various methods of determiningT_(m) values may produce slightly differing values for the same DNAmolecule, but those values typically do not vary from each other by morethan about 2° C. or 3° C.

In other embodiments, the T_(m) values can be calculated using knownmethods for predicting oligonucleotide melting temperatures (see, e.g.,SantaLucia, 1998, Proc. Natl. Acad. Sci. USA 95:1460-1465; Frier et al.,1986, Proc. Natl. Acad. Sci. USA 83:9373-9377; Breslauer, 1986, Proc.Natl. Acad, Sci. USA 83:3746-3750; Rychlik et al., 1989, Nucleic AcidsRes. 17:8543-8551; Rychlik et al., 1989, Nucleic Acids Res.18:6409-6412; Wetmur, 1991, Crit. Rev. Biochem. Mol. Biol. 26:227-259;Osborne, 1991, CABIOS 8:83; Montpetit et al., 1992, J. Virol. Methods36:119-128).

Like amplification primers for conventional PCR or RT-PCR, the sequencesof amplification primer pairs useful for multiplex amplifications aredesigned to be substantially complementary to regions of the targetpolynucleotides that flank the sequence of interest to be amplified. By“substantially complementary” is meant that the sequences of the primersinclude enough complementarity to hybridize to the targetpolynucleotides under the temperature and conditions employed in themultiplex amplification reaction.

Although in many instances the sequences of the primers may becompletely complementary to the template polynucleotide, in someinstances it may be desirable to include regions of mis-match ornon-complementarity, as is well known in the art. As a specific example,a region of non-complementarity may be included at the 5′-end of one ormore of the primers, with the remainder of the primer sequences beingcompletely complementary to their respective target polynucleotidesequences. As another specific example, non-complementary bases orlonger regions of non-complementarity can be interspersed throughout theprimer, provided that the primer has sufficient complementarity tohybridize to the target polynucleotide sequence under the temperaturesand reaction conditions used for the multiplex amplification.

One or more of the primers can include a label, e.g., at the 5′terminus. The term “label” refers to any moiety that, when attached tocompounds such as polynucleotides, render such compounds detectableusing known detection means, e.g., spectroscopic, photochemical,radioactive, biochemical, immunochemical, enzymatic or chemical means.Exemplary labels include but are not limited to fluorophores,chromophores, radioisotopes, spin labels, enzyme labels, infraredlabels, and chemiluminescent labels. Other examples of labels includemembers of conventional binding pairs, such as biotin, which may be usedwith avidin in a capture method or for further concentrating the sample.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Means of detecting such labels are well known to those ofskill in the art.

In certain embodiments, primers can also include a 5′ “tail” (see, e.g.,Bengra et al., 2002, Clin. Chem. 48:2131-2140 and Myakishev et al.,2001, Genome Res. 11:163-169) for universal amplification, detectionand/or purification. In some embodiments, primers can include a tagportion for binding to a tag-complement portion of a mobility modifier(see, e.g., U.S. Pat. No. 6,395,486 to Grossman). Exemplary tags and/ortag complements include but are not limited to antibodies and associateantigen or hapten, receptors and associated ligands, avidin (orstreptavidin) and biotin, and polynucleotide sequences and theircomplementary sequences. A mobility modifier typically also includes atail portion, such as a polymer, for effecting a particular mobility amobility-dependent analysis technique.

The chemical composition of the amplification primers is not critical tothe success of the multiplex amplifications described herein. The onlyrequirement is that the DNA polymerase used in the multiplexamplification reaction be able to extend the primers when hybridized toa target polynucleotide. A variety of oligonucleotides that are capableof being extended by DNA polymerases in template-dependent primerextension reactions are known in the art. Examples of theseoligonucleotides include, but are not limited to, DNA, RNA, PNA and LNAoligonucleotides, or various combinations and/or chimeras thereof. Forexample, a chimeric oligonucleotide can comprise a region of DNA fusedto a region of, for example, RNA, PNA or LNA. As a specific example, achimeric amplification primer may include two regions of PNA linked by a2′-deoxyribonucleotide or region of DNA (see, e.g., Internationalpublication No. WO/9640709, incorporated herein by reference, formethods and compositions related to PNA/DNA chimera preparation). Theamplification primers may be wholly composed of the standardgene-encoding nucleobases (e.g., cytidine, adenine, guanine, thymine anduracil) or, alternatively, they may include modified nucleobases knownby skilled artisans to form base-pairs with the standard nucleobases andto be extendible by polymerases when included in primers. Specificexamples of such modified nucleobases include, but are not limited to,7-deazaguanine and 7-deazaadenine. Other suitable modified ornon-standard nucleobases will be apparent to those of skill in the art.

In addition, the amplification primers may include one or more modifiedinterlinkages, such as one or more phosphorothioate orphosphorodithioate interlinkages, as is well-known in the art.

All of these types of various oligonucleotides, or mixtures of sucholigonucleotides, may be used as amplification primers in the variousmultiplex amplifications described herein. In one embodiment, all of theprimers used in the multiplex amplification reaction are DNAoligonucleotides.

The number of different amplification primer pairs utilized in themultiplex amplification is not critical and can range from as few astwo, to as many as tens, hundreds, thousands, or even more. Thus,depending upon the particular application and conditions, the multiplexamplifications permit the simultaneous amplification of from as few astwo, to as many as tens, hundreds, thousands, or even more,polynucleotide sequences of interest.

As will be described in more detail below, the product of a multiplexamplification can be used in a variety of different downstream assaysand/or analyses. In a specific embodiment that will be discussedfurther, below, the product of a multiplex amplification reaction may beused in a plurality of subsequent, single-plex (“simplex”), quantitativeor real-time PCR amplification reactions, such as for example, thequantitative or real-time amplifications routinely employed for geneexpression analysis and which are commonly known in the art as5′-exonuclease assays or TaqMan® assays (see, e.g., U.S. Pat. No.5,691,146). When the product of the multiplex amplification reaction isto be used in this manner, the number of and/or sequences of theamplification primer pairs utilized in the multiplex amplificationreaction can be correlated to correspond to the number of downstreamsingle-plex, quantitative amplification reactions that may be performed.For example, if 96 downstream single-plex 5′-exonuclease amplificationassays are desired, then the multiplex amplification can be carried outwith a pool of 96 different sets of amplification primers or pairs.Correlating the number of amplification primer pairs with the number ofsubsequent single-plex quantitative amplification reactions isparticularly convenient or advantageous in embodiments in which eachsubsequent single-plex quantitative amplification reaction will becarried out with a pair of amplification primers identical in sequenceto one of the pairs used in the multiplex amplification reaction.

The number and/or sequences of the amplification primer pairs used for amultiplex amplification reaction can be correlated in a similar mannerto other downstream assays or analyses that may be performed with theproduct of the multiplex amplification reaction. For example, inembodiments where the product of the multiplex amplification reactionwill be used for gene expression analysis on, for example, anoligonucleotide array, the multiplex amplification primer pairs can bedesigned to specifically amplify the polynucleotide sequences that willbe assessed by the microarray.

The sequences of multiplex primer pairs suitable for generatingmultiplex amplification product suitable for use in particular desiredsubsequent analyses and/or assays will be apparent to those of skill inthe art.

As discussed above, depending upon the nature of the samplepolynucleotides to be amplified (e.g., RNA or DNA), a multiplexamplification reaction can be accomplished by polymerase chain reaction(PCR) or reverse-transcription PCR (RT-PCR). Thus, multiplexamplifications in which the target polynucleotide(s) is a DNA willtypically include as essential components, in addition to the pluralityof amplification primer pairs or sets discussed above, a mixture of2′-deoxribonucleoside triphosphates suitable for template-dependent DNAsynthesis (e.g., primer extension) and a DNA polymerase. Multiplexamplifications in which the target polynucleotide(s) is a RNA willtypically additionally include a reverse-transcriptase. With theexception of certain parameters described below, and the use of aplurality of amplification primer pairs instead of a single pair asdescribed above, the multiplex amplification reactions may be carriedout using reagents, reagent concentrations and reaction conditionsconventionally employed in such conventional PCR and RT-PCR reactions.For example, except as noted herein, the various different primerconcentrations, enzymes (e.g. DNA polymerases and reversetranscriptases), enzyme concentrations, dNTP mixtures (as well as theirabsolute and/or relative concentrations), total target polynucleotideconcentrations, buffers, buffer concentrations, pH ranges, cycling timesand cycling temperatures employed in conventional PCR and RT-PCRreactions may be used for the multiplex amplification reactionsdescribed herein. Guidance for selecting suitable reaction conditionsmay be found, for example, in U.S. Pat. Nos. 4,683,202; 4,683,195;4,800,159; 4,965,188; 5,561,058; 5,618,703; 5,693,517; 5,876,978;6,087,098; 6,436,677; and 6,485,917, and PCR Essential Data, J. W. Wiley& Sons, Ed. C. R. Newton, 1995, and PCR Protocols: A Guide to Methodsand Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T.,eds.), Academic Press, San Diego (1990), all of which are incorporatedherein by reference. A variety of tools for designing PCR and RT-PCRamplification primers, as well as myriad protocols, reaction conditionsand techniques for carrying out various different types of PCRreactions, including conventional PCR reactions and RT-PCR reactions arealso available online (see, e.g.,http://www.protocol-online.org/prot/Molecular_Biology/PCR/index.html).All of these various tools and protocols can be used in connection withthe multiplex amplification reactions described herein.

Like conventional PCR and RT-PCR amplification reactions, the multiplexamplification reactions may be carried out with a variety of differentDNA polymerases (or mixture of DNA polymerases), but are preferablycarried out in the presence of one or more thermostable polymerases.Suitable thermostable polymerases include, but are not limited to, Taqand Tth (commercially available from Applied Biosystems, an AppleraCorporation business). Moreover, like conventional RT-PCR amplificationreactions, multiplex RT-PCR amplification reactions may be carried outwith a variety of different reverse transcriptases (or mixture ofreverse transcriptases), although in some embodiments thermostablereverse-transcriptions are preferred. Suitable thermostable reversetranscriptases include, but are not limited to, reverse transcriptasessuch as AMV reverse transcriptase, MuLV, and Tth reverse transcriptase.Temperatures suitable for carrying out the various denaturation,annealing and primer extension reactions with the polymerases andreverse transcriptases are well-known in the art. Optional reagentscommonly employed in conventional PCR and RT-PCR amplificationreactions, such as reagents designed to enhance PCR, modify T_(m), orreduce primer-dimer formation, may also be employed in the multiplexamplification reactions (see e.g., U.S. Pat. Nos. 6,410,231; 6,482,588;6,485,903; and 6,485,944, all of which are incorporated herein byreference). In certain embodiments, the multiplex amplifications may becarried out with commercially-available amplification reagents, such as,for example, AmpliTaq® Gold PCR Master Mix, TaqMan® Universal Master Mixand TaqMan® Universal Master Mix No AmpErase® UNG, all of which areavailable commercially from Applied Biosystems, an Applera Corporationbusiness.

Although suitable results can be achieved using conventional reagentsand reaction conditions, in many embodiments the performance or observedefficiency of a multiplex amplification can be improved by adjustingcertain parameters and/or conditions of the reaction.

The expressions “observed performance” and “observed efficiency” andgrammatical equivalents thereof when used in connection with multiplexamplifications refer to the amounts of the individual ampliconsgenerated in the multiplex amplification. Amplification performance orefficiency can correspond to a multiplex amplification generally, or toa specific amplicon within a multiplex amplification. A multiplexamplification is 100% efficient for a specific amplicon of interest whenthe quantity of the specified amplicon generated in the multiplexamplification is identical to that produced in a single-plex,conventional PCR or RT-PCR reaction with the same target polynucleotide.A multiplex reaction is 100% efficient generally when the amounts ofeach of the amplicons produced in the multiplex amplification areidentical to the amounts of the respective amplicons produced inindividual, single-plex conventional PCR or RT-PCR reactions with thesame target polynucleotides. Multiplex amplifications are considered“highly efficient” for a specific amplicon when the amount of thespecified amplicon generated in a multiplex reaction is within about≥90% of the amount generated in a single-plex, conventional PCR orRT-PCR reaction.

The observed efficiency of a multiplex amplification reaction, eithergenerally or with respect to a specified target sequence, can beassessed using real-time PCR methods. In one embodiment, the observedefficiency of amplification of a specific target sequence can bedetermined by amplifying a plurality of polynucleotides, which includesthe specific target, in a multiplex PCR amplification reaction using aplurality of primer sets, each of which is suitable for amplifying adifferent target sequence of interest. The multiplex amplification iscarried out for N thermal cycle steps, where N can be selected by theuser. A first aliquot of multiplex amplification product is thenamplified in a single-plex PCR amplification reaction in the presence ofan amplification primer pair or set suitable for amplifying the targetsequence of interest and a reagent useful for monitoring theamplification reaction in real time, such as an intercalating dye or asequence-specific oligonucleotide probe (e.g., a TaqMan probe). A secondaliquot obtained from a “mock” (control) multiplex amplification thatwas not subjected to thermal cycling is similarly single-plex amplified.A Ct^(assay) value is obtained from the first aliquot and a Ct^(control)value is obtained from the second aliquot. In one embodiment, theobserved efficiency of the amplification of the specified targetsequence in the multiplex amplification can be calculated from thefollowing equation:% observed efficiency=100×(Ct ^(control) value−Ct ^(assay) value)/N

Any number of specific target sequences in a multiplex amplification canbe similarly analyzed. A user can select a selection criteria (i.e. a“cut-off” value) for the observed efficiency such as, for example, 50%,70%, 80%, 90%, 95% or 99%. If desired, the observed efficiencies foreach primer set in a multiplex amplification can be determined and theprimers grouped according to whether their observed efficiencies equalor exceed the selection criteria. Primer sets that do not meet or exceedthe selection criteria can be analyzed individually in singleplexamplifications, or can be re-grouped into one or more separate pools ofprimer sets for further analysis.

In another embodiment, the average observed efficiency of amplificationof all of the target sequences in a multiplex amplification can beanalyzed. As for the embodiment discussed above, a multiplexamplification can be carried out for N thermal cycles, the exact numberof which can be selected by the user. The product of the multiplexamplification is divided into a plurality of aliquots, typically into anumber of aliquots equal to the number of primer pairs used in themultiplex amplification. Each aliquot is single-plex amplified in thepresence of one of the sets of primer pair used in the multiplexamplification and a reagent or probe suitable for monitoring thesingle-plex amplification real time. A Ct^(assay) value is determinedfor each single-plex amplification and an average Ct^(assay) valuecalculated therefrom. Ct^(control) values and an average Ct^(control)value are similarly obtained from single-plex amplifications of a “mock”(control) multiplex amplification reaction that was not subjected tothermal cycling. In one embodiment, the average efficiency of themultiplex amplification can be calculated using the following equation:% average efficiency=100×(average Ct ^(control) value−average Ct^(assay) value)/N

As will be recognized by skilled artisans, a particular degree ofefficiency for a multiplex amplification generally, or for a specificsequence or sequences, is not required for success. All that is requiredis that the multiplex amplification perform in a manner suitable for aparticular application. As discussed above, in many embodiments suitablyefficient multiplex amplifications are achieved using conventional PCRor RT-PCR reaction conditions. However, it has been discovered that theefficiency of multiplex amplifications can be improved beyond thatachieved using conventional PCR or RT-PCR reaction conditions bymodifying certain of the reaction conditions or parameters, such as thequantity of DNA polymerase used.

Typically, conventional PCR and RT-PCR reactions are carried out with0.05 U/μL DNA polymerase. For a 10 cycle 95-plex amplification carriedout with 1 U/20 μl. AmpliTaq Gold DNA polymerase (Applied Biosystems, anApplera Corporation business), it has been found that adding anadditional 1-8 U/20 μL increases the efficiency of the multiplexamplification. As will be described in more detail in the Examplessection, significant increases in efficiency were observed with anadditional 1-5 U/20 μl, AmpliTaq Gold DNA polymerase. Increases werealso observed with an additional 6-15 U/204, but they were lesspronounced, potentially, due to the additional glycerol added to thereaction mixture as a result of spiking the reaction mixture withAmpliTaq® Gold DNA polymerase stored in 50% glycerol. (see, e.g., FIG.3). Similar increases in efficiency are also expected for other DNApolymerases, such as, for example, TaqI polymerase, Klenow fragment ofDNA polymerase I, SEQUENASE 1.0 and SEQUENASE 2.0 (U.S. Biochemical), T5DNA polymerase and Phi29 DNA polymerase. Thus, in one embodiment, amultiplex amplification is carried out in the presence of from about 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 Units DNA polymeraseper every 20 μL of reaction volume.

The efficiency of a multiplex amplification reaction may also beincreased by employing a longer duration for the primer extensionreaction than is typically used in conventional PCR or RT-PCR, eitheralone or in combination with the increased amount of DNA polymerasediscussed above. In conventional PCR and RT-PCR reactions, the primerextension reaction is typically carried out for approximately 1 min. peramplification cycle. While not intending to be bound by any particulartheory of operation, it is believed that increasing the duration of theprimer extension reaction from 1 min. to, for example, about 2, 3, 4, 5,6, 7, 8, 9, 10 min. or even longer, may improve the performance orefficiency of a multiplex amplification. Thus, in one embodiment, amultiplex amplification is carried out using a duration for the primerextension reaction in the range of about 2-15 min. per cycle. Thedurations used for the other intervals making up a single reaction cyclecan be those used conventionally. In one embodiment, a multiplexamplification may be carried out using a two-step cycle including afirst denaturation step of 95° C. for 15 seconds and a secondanneal/extend step of 60° C. for 1 to 15 minutes.

Conventional PCR and RT-PCR employ concentrations of amplificationprimers in the range of about 300-900 nM each primer. Although primerconcentrations in this range can be used in the multiplexamplifications, it has been discovered that multiplex amplifications maybe carried out using considerably lower amplification primerconcentrations. Quite surprisingly, in 95-plex amplifications carriedout for 10 cycles using a primer extension reaction time of 10min/cycle, highly efficient multiplex amplification was achieved withprimer concentrations as low as 45 nM each primer. In certainembodiments, primer concentrations in the range of about 30-45 nM eachprimer may be used.

Moreover, it was discovered that it is not necessary to optimize theconcentrations of the individual primer pairs. Regardless of thesequences being amplified, and hence the sequences of the primers, theamplification primers can be used at concentrations in the range ofabout 30-900 nM each primer. Different amplification primer pairs may bepresent at different concentrations within this range or, alternatively,some or all of the amplification primers may be present at approximatelyequimolar concentrations within this range. In one embodiment, at leastsome of the amplification primers, for example, approx. 10%, 25%, 35%,50%, 60%, or more, are present in approximately equimolar concentrationsranging from about 30 nM to about 100 nM each primer. In anotherembodiment, all of the amplification primers are present atapproximately equimolar concentrations in the range of about 30 nM to100 nM each primer. In certain embodiments, all of the amplificationprimers are present at concentrations of 30, 40, 45, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800 or 900 nM each primer. In yetanother embodiment, some or all of the amplification primers are presentin a concentration of about 45 nM each primer. The amplification primerconcentrations discussed above can be used regardless of whether thetarget polynucleotide(s) being multiplex amplified are RNA or DNA. Thereverse-transcription reaction of a multiplex RT-PCR amplification workswell at the stated primer concentrations.

As will be recognized by skilled artisans, PCR and RT-PCR reactions canbe broken up into three phases: an exponential phase in which the amountof amplicon accumulates exponentially every cycle (i.e., doubles everycycle); a linear phase in which the amount of amplicon accumulates at avariable rate every cycle (i.e., the reaction begins to slow); and aplateau phase, where the reaction has stopped, no more amplicon is beingproduced and, if left long enough, the amplicon will begin to degrade.

In the exponential phase, the degree of amplification achieved isexponentially proportional to the number of amplification cyclesemployed. For example, a 10-cycle amplification yields a 1024-foldincrease in the quantity of amplified sequence. As another example, a15-cycle amplification yields a 32,286-fold increase. In anotherexample, a 20-cycle amplification yields a 1,048,576-fold increase.

The number of amplification cycles performed with a multiplexamplification may depend upon, among other factors, the degree ofamplification desired. The degree of amplification desired, in turn, maydepend upon such factors as the amount of polynucleotide sample to beamplified and/or the intended downstream use of the multiplexamplification product. Accordingly, the number of cycles employed in amultiplex amplification will vary for different applications and will beapparent to those of skill in the art. For most applications, reactionscarried out for 10 amplification cycles are expected to yield sufficientmultiplex amplification product for several hundred downstream analyses,even when the sample is derived from 1 to a few cells, is present invery low copy number, and/or is present only as a single copy,regardless of the amount of sample required to perform the analysis.However, more or fewer amplification cycles may be employed. In certainembodiments the multiplex amplification is carried out for as many as10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more cycles. In specificembodiments, the multiplex amplification is carried out for 2-12 cycles,inclusive, for 5-11 cycles, inclusive, or for up to 14 cycles (e.g., seeExample 7). A high number of cycles may be required in certainapplications where a large plurality of downstream assays are to beperformed.

In many embodiments, it may be desirable to keep the multiplexamplification from progressing beyond the exponential phase or thelinear phase. Indeed, in many embodiments, it may be desirable to carryout the multiplex amplification for a number of cycles suitable to keepthe reaction within the exponential or linear phase.

Multiplex amplification reaction parameters (e.g., concentrations ofprimer pairs or sets, concentration of DNA polymerase, duration andannealing, primer extension and/or denaturation steps, number of thermalcycles, Mg²⁺ concentration, concentration of adjuvants (such as DMSO,glycerol, BSA or urea) can be optimized empirically, for example bycarrying out a plurality of multiplex amplifications varying one (ormore) of these parameters and assessing the efficiencies of theresultant multiplex amplifications, for example using the methodsdescribed above. An example of such an optimization reaction, in whichthe amount of DNA polymerase was varied, is described in Example 1,below.

Once amplified, the multiplex amplification product can be used inmyriad different subsequent assays or analyses without furtherpurification or manipulation. For such subsequent assays or analyses,the product of the multiplex amplification can be divided, either withor without prior dilution, amongst a plurality of different assays oranalyses. The degree of any optional dilution may depend upon suchfactors as the number of subsequent analyses desired and the amount ofsample required for each such analysis, and will be apparent to those ofskill in the art. Examples of subsequent assays and analyses that can becarried out with a multiplex amplification product, but are not limitedto, single nucleotide polymorphism (SNP) analysis, genotyping analysis,gene expression analysis (e.g., via quantitative PCR or RT-PCR or viahybridization on oligonucleotide microassays), finger printing analysis,nucleic acid sequencing (e.g., U.S. Pat. No. 6,428,986), nucleic acidmini-sequencing (e.g., U.S. Pat. No. 6,479,242), and/or for hybridizingto arrays that may be used in gene expression profiling (e.g., U.S. Pat.No. 6,485,944).

In certain embodiments, the product of the multiplex amplification isnot divided amongst a plurality of different assays or analyses. Forexample, one or more amplification primers, probes, dyes and/or otherreagents may be added directly to the amplification product in order tocarry out additional amplifications. In various other embodiments, theproduct of the multiplex amplification is divided amongst a plurality ofaliquots, and one or more of these aliquots can be subject to amultiplex assay or analysis, examples of which include multiplexamplification and multiplex SNP detection.

In one specific embodiment, the product of the multiplex amplificationmay be used in subsequent amplification reactions, such as thequantitative or real-time amplification assays commonly used for geneexpression analysis. In a specific example of such a quantitative orreal-time amplification assays, total RNA from a sample is amplified byRT-PCR in the presence of amplification primers suitable forspecifically amplifying a specified gene sequence of interest and anoligonucleotide probe labeled with a labeling system that permitsmonitoring, for example via the 5′-exonuclease activity of the DNApolymerase employed in the RT-PCR amplification, of the quantity ofamplicon that accumulates in the amplification reaction in real-time.The cycle threshold values (Ct values) obtained in such quantitativeRT-PCR amplification reactions can be correlated with the number of genecopies present in the original total mRNA sample. Such quantitative orreal-time RT-PCR reactions, as well as different types of reagentsand/or labeled oligonucleotide probes useful for monitoring theamplification in real time, are well-known in the art. A specific assaywhich utilizes the 5′-exonuclease assay to monitor the amplification asa function of time is referred to as the 5′-exonuclease genequantification assay (see, e.g., U.S. Pat. Nos. 5,210,015 and 5,538,848and Lie & Petropoulos, 1998, Curr. Opin. Biotechnol. 14:303-308).Another specific assay, which utilizes intercalating or other dyes tomonitor an amplification as a function of time, is described in U.S.Pat. No. 5,994,056.

Although powerful, 5′-exonuclease gene quantification assays (as well asother gene quantification assays, such as quantification assaysperformed on DNA microarrays) require relatively large amounts ofstarting RNA (e.g., from 1-10 μg). Owing to this large samplerequirement, 5′exonuclease gene quantification assays and other genequantification assays have not been suitable for detecting genesexpressed at low copy numbers, or in instances where only limitedquantities of sample is available (e.g., from clinical biopsies, etc.).

By virtue of the ability to amplify simultaneously a plurality ofpolynucleotide sequences in a sample, the multiplex amplificationreactions described herein are ideal for use in connection with the suchdownstream gene expression analyses, such as, for example the5′-exonuclease gene quantification assay. Polynucleotides present insamples at extremely low copy numbers, and/or samples obtained from afew or even a single cell, may be multiplex amplified so as to provideamounts of sample suitable for tens, hundreds or even thousands ofquantitative or real-time amplification assays. Accordingly, in oneembodiment of the invention, the product of the multiplex amplificationreaction is divided, either with or without prior dilution, amongst aplurality of single-plex quantitative or real-time amplificationreactions. Each single-plex quantitative or real-time amplificationreaction is carried out in a conventional manner with a single set ofamplification primers and a suitable probe. The amplification primerpair or set used for the single-plex quantitative or real-timeamplification can be the same as one of the primer pairs or sets used inthe multiplex amplification reaction. Significantly, the multiplexamplification product can be used directly in such subsequentsingle-plex amplifications without further purification or manipulation.The various enzymes, dNTPs, amplification primers and other optionalreagents carried over from the multiplex amplification do not interferewith the accuracy of the subsequent quantitative or real-timeamplification assays.

The present inventor has surprisingly discovered, in certainembodiments, that the multiplex amplification substantially maintainsthe copy number ratios, presumably due to high efficiency ofamplification, so that the copy numbers or expression levels of theoriginal sample can be ascertained from the multiplex amplified sample.In some embodiments, relative copy numbers from the original sample canthus be determined and used, for example, in various downstreamapplications, such as gene expression studies.

Samples amplified in a multiplex fashion may be used in a wide varietyof subsequent analysis or assay without further purification ormanipulation. For example, the product of the multiplex amplificationmay be used for single polynucleotide polymorphism (“SNP”) analysis,genotyping analysis, gene expression analysis, fingerprinting analysis,analysis of gene mutations for genetic diagnoses, analysis of rareexpressed genes in cells, nucleic acid sequencing (e.g., U.S. Pat. No.6,428,986), and nucleic acid mini-sequencing (e.g., U.S. Pat. No.6,479,242).

In performing gene expression studies, for example, various methods forquantifying a polynucleotide product in the multiplex amplified samplecan be used. The term “quantifying” when used in the context ofquantifying transcription levels of a gene can refer to absolute or torelative quantification. Absolute quantification may be accomplished byinclusion of known concentration(s) of one or more target nucleic acids(e.g. control nucleic acids or with known amounts the target nucleicacids themselves) and referencing the detected signal of unknowns withthe known target nucleic acids (e.g. through generation of a standardcurve). Alternatively, relative quantification can be accomplished bycomparison of detected signals between two or more genes, or between twoor more treatments to quantify the changes in detected signal valuesand, by implication, transcription level. The detected signal willdepend upon the particular method utilized. For example, when usingreal-time PCR, the detected signal can be related to a fluorescenceintensity. Amplification products can be separated and detected by anyof a variety of techniques known to those of skill in the art (see,e.g., U.S. Pat. No. 6,618,679). The data obtained from the detection canbe stored and analyzed to obtain a set of gene expression data. Whenusing a microfabricated DNA array, the detected signal can be ahybridization intensity.

In particular embodiments, the product of a multiplex amplification canbe applied to solid supports containing polynucleotide hybridizationprobes for differentially expressed genes. Any solid surface to whichpolynucleotides can be bound, either directly or indirectly, eithercovalently or non-covalently, can be used. Non-limiting examples of suchsupports include filters, polyvinyl chloride dishes, beads, glass slidesetc. A particular example of a solid support is a high density array orDNA chip. These contain a particular hybridization probe in apredetermined location on the array. In some embodiments, eachpredetermined location may contain more than one molecule of the probe,but each molecule within the predetermined location has an identicalsequence. Such predetermined locations are termed features. There maybe, for example, from 2, 10, 100, 1000, to 10,000, 100,000, or 400,000of such features on a single solid support. The solid support, or thearea within which the probes are attached may be on the order of asquare centimeter. Hybridization probe arrays for expression monitoringcan be made and used according to any techniques known in the art. (Seefor example, Lockhart, D. J. et al., 1996, Nature Biotechnology14:1675-1680; McGall, G. et al., 1996, Proc. Nat. Acad. Sci. USA93:13555-13460; and U.S. Pat. Nos. 6,033,860, 6,309,822, 6,485,944, and6,548,257).

The present inventor has surprisingly discovered that the presence ofconventional concentrations of oligonucleotide probes, such as5′-exonuclease probes, in a multiplex amplification reaction does notinterfere with the performance or efficiency of the multiplexamplification. Nor does the presence of such probes interfere withdownstream analyses, such as single-plex quantitative or real-timeamplification assays or other analyses, carried out with the product ofthe multiplex amplification. This discovery permits multiplexamplifications to be carried out using commercially-available,off-the-shelf quantitative or real-time amplification reagents, such asthe Assays-On-Demand reagents commercially available from AppliedBiosystems (an Applera Corporation business).

The ability to carry out a multiplex amplification with commerciallyavailable Assays-On-Demand 5′-exonuclease reagents (or othercommercially-available reagents) permits the creation of multiplexamplification reactions that are ideally correlated or matched withsubsequent single-plex 5′-exonuclease assays. By correlated or matchedis meant that the same sets of primers or primer pairs that are used inthe multiplex amplification step may be used in the downstreamanalytical assays. However, in some embodiments, the primers or primerpairs used in the downstream assays may be different from the primerpairs used in the upstream multiplex amplification. In certainembodiments, the primers used in a the downstream assays may or may notbe nested primers.

Quite advantageously, kits suitable for carrying out a multiplexamplification followed by a plurality of single-plex quantitative orreal-time amplification assays can be readily created fromreadily-available 5′-exonuclease reagents without requiring additionalmanipulations or purification. The primers for performing the multiplexamplification can be created by pooling together 5′-exonuclease reagentscomprising a pair of amplification primers and a 5′-exonuclease probe.As mentioned above, the presence of the 5′-exonuclease probes in themultiplex amplification reaction and subsequent single-plex5′-exonuclease amplification assays does not interfere with eitheramplification.

An embodiment of a matched or correlated multi-step assay, which can becreated using the Assays-On-Demand® service available from AppliedBiosystems (see e.g.,http://www.appliedbiosystems.com/products/productdetail.cfrn?prod_id=1101) is illustrated in FIG. 2. Referring to FIG. 2, a pluralityof 5′-exonuclease amplification primer/probe sets are selected by theuser and pooled together to yield a plurality of amplification primerpairs or sets suitable for multiplex amplification (the pool alsoincludes the plurality of 5′-exonuclease probes). A separate aliquot ofeach of the selected 5′-exonuclease amplification primer/probe sets aredispensed into individual reaction vessels, such as the wells of amultiwell plate, a single primer/probe set per vessel or well. In afirst step, target polynucleotides from a sample of interest aremultiplex amplified in the presence of the pooled amplification5′-exonuclease primers/probes. The product of the multiplexamplification is then aliquoted into the wells of the multiwell plate,and single-plex 5′-exonuclease amplification assays are carried outusing conventional methods. In one particularly convenient embodiment,the 5′-exonuclease primer/probe sets may be dispensed among the wells ofa micro fluidic card that can be used directly on an instrument designedfor quantitative or real-time amplification analysis, such as the ABPrism 7900 HT instrument available from Applied Biosystems (an AppleraCorporation business). An example of a suitable microcard is describedin U.S. Pat. No. 6,126,899 and a commercial embodiment is the 7900HTMicro Fluidic card available from Applied Biosystems (an AppleraCorporation business).

Oligonucleotide probes that can be present in the multiplexamplification are not limited to 5′-exonuclease probes. In oneembodiment, any labeled or unlabeled single-stranded oligonucleotidewhich is complementary to all or part of an amplified target sequence,may be present in the multiplex amplification.

Like the primers discussed above, such oligonucleotide probes may beDNA, RNA, PNA, LNA or chimeras composed of one or more combinationsthereof. The oligonucleotides may be composed of standard ornon-standard nucleobases or mixtures thereof and may include one or moremodified interlinkages, as previously described in connection with theamplification primers. The oligonucleotide probes may be suitable for avariety of purposes, such as, for example to monitor the amount of anamplicon produced, to detect single nucleotide polymorphisms, or otherapplications as are well-known in the art.

In one embodiment, each oligonucleotide probe is complementary to atleast a region of a specified amplicon. The probes can be completelycomplementary to the region of the specified amplicons, or they may besubstantially complementary thereto (at least about 65% complementaryover a stretch of at least 15 to 75 nucleotides). In other embodiments,the probes are at least about 75%, 85%, 90%, or 95% complementary to theregions of the amplicons. See Kanehisa, M., 1984, Nucleic Acids Res. 12:203, incorporated herein by reference. The exact degree ofcomplementarity between a specified oligonucleotide probe and ampliconwill depend upon the desired application for the probe and will beapparent to those of skill in the art.

The lengths of such oligonucleotide probes can vary broadly, and in someembodiments can range from as few as two as many as tens or hundreds ofnucleotides, depending upon the particular application for which theprobe was designed. In one specific embodiment, the oligonucleotideprobes range in length from about 15 to 35 nucleotides. In anotherspecific embodiment, the oligonucleotide probes range in length fromabout 15 to 25 nucleotides. In yet another specific embodiment,oligonucleotide probes can range from 25 to 75 nucleotides. In otherembodiments, the probes range in length from about 6 to 75 nucleotidesor from about 12 to 22 nucleotides. An oligonucleotide probe can includea 5′ tag portion for binding with a mobility modifier (e.g., asdescribed in U.S. Pat. No. 6,395,486).

In one specific embodiment, oligonucleotide probes present in amultiplex amplification are suitable for monitoring the amount ofamplicon(s) produced as a function of time. Such oligonucleotide probesinclude, but are not limited to, the 5′-exonuclease assay (TaqMan®)probes described above (see also U.S. Pat. No. 5,538,848), variousstem-loop molecular beacons (see, e.g., U.S. Pat. Nos. 6,103,476 and5,925,517 and Tyagi & Kramer, 1996, Nature Biotechnology 14:303-308),stemless or linear beacons (see, e.g., WO 99/21881), PNA MolecularBeacons™ (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNAbeacons (see, e.g., Kubista et al., 2001, SPIE 4264:53-58), non-FRETprobes (see, e.g., U.S. Pat. No. 6,150,097), Sunrise®/Amplifluor® probes(U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion™ probes(Solinas et al., 2001, Nucleic Acids res. 29:E96 and U.S. Pat. No.6,589,743), bulge loop probes (U.S. Pat. No. 6,590,091), pseudo knotprobes (U.S. Pat. No. 6,548,250), cyclicons (U.S. Pat. No. 6,383,752),MGB Eclipse™ probe (Epoch Biosciences), hairpin probes (U.S. Pat. No.6,596,490), peptide nucleic acid (PNA) light-up probes, self-assemblednanoparticle probes, and ferrocene-modified probes described, forexample, in U.S. Pat. No. 6,485,901; Mhlanga et al., 2001, Methods25:463-471; Whitcombe et al., 1999, Nat. Biotechnol. 17:804-807;Isacsson et al., 2000, Mol. Cell. Probes. 14:321-328; Svanvik et al.,2000, Anal Biochem. 281:26-35; Wolffs et al., 2001, Biotechniques766:769-771; Tsourkas et al., 2002, Nucleic Acids Res. 30:4208-4215;Riccelli et al., 2002, Nucleic Acids Res. 30:4088-4093; Zhang et al.,2002, Shanghai. 34:329-332; Maxwell et al., 2002, J. Am. Chem. Soc.124:9606-9612; Broude et al., 2002, Trends Biotechnol. 20:249-56; Huanget al., 2002, Chem. Res. Toxicol. 15:118-126; and Yu et al., 2001, J.Am. Chem. Soc. 14:11155-11161, all of which are incorporated herein byreference.

In another embodiment, the oligonucleotide probes are suitable fordetecting single nucleotide polymorphisms, as is well-known in the art.A specific example of such probes includes a set of four oligonucleotideprobes which are identical in sequence save for one nucleotide position.Each of the four probes includes a different nucleotide (A, G, C andT/U) at this position. The probes may be labeled with labels capable ofproducing different, detectable signals that are distinguishable fromone another, such as different fluorophores capable of emitting light atdifferent, spectrally-resolvable wavelengths (e.g., 4-differentlycolored fluorophores). Such labeled probes are known in the art anddescribed, for example, in U.S. Pat. No. 6,140,054 and Saiki et al.,1986, Nature 324:163-166.

Performing multiplex amplification in the presence of these variousoligonucleotide probes permits a great deal of flexibility in designingor creating sets of amplification primers for multiplex amplifications.Commercially available primer sets including such oligonucleotide probescan be simply pooled together without prior removal of theoligonucleotide probes and used for multiplex amplification withoutfurther manipulation.

In some embodiments, the oligonucleotide probes can be removed frompooled primer sets prior to multiplex amplification. Such removal can beeffected using pairs of specific binding molecules, such asbiotin/avidin or antibody/antigen. For example, a biotin-labeledoligonucleotide probe can be removed by avidin binding. In otherembodiments, labeled nucleotide probes can be photobleached using laseror other light sources.

The multiplex amplification may also be carried out in the presence ofdye molecules suitable for, for example, monitoring the accumulation ofamplification products at the end of the amplification and/or during theamplification as a function of time. In one embodiment, such dyesinclude dyes that produce a detectable signal, such a fluorescence, whenbound to double-stranded polynucleotides. Non-limiting examples ofsuitable dyes include common nucleic acid stains, such as intercalatingdyes and minor groove binding dyes, as are well-known in the art. In aspecific embodiment, the dye is SYBR® Green I or II, ethidium bromide,or YO-PRO-1 (available from Molecular Probes, Eugene, Oreg.). Such dyescan be used at conventional concentrations commonly employed in the art(see, e.g., U.S. Pat. No. 5,994,056).

In carrying out a multiplex amplification in the presence of such a dyemolecule, or in the presence of suitable oligonucleotide probes,Applicants have discovered a general method for characterizing pooledsets of primers. In the method, the amplification is monitored in realtime, and a cycle threshold value (“Ct^(pool)”) obtained. This is anadditive signal produced by the summation of all of the amplicons. Inthe embodiment in which oligonucleotide probes are used for the realtime multiplex amplification, a separate probe is present for eachtarget sequence being amplified, and all of the probes use the samesignaling system. The method is especially useful in providing a rapidand convenient test of pooled reagents that may be provided inready-made, pre-optimized, kits. Such a pool of reagents can be preparedby mixing commercially available primer sets such as theAssays-on-Demand™ Gene Expression products or the primer sets availablein the QuantiTect Gene Expression Assays (Qiagen), as further describedherein.

In certain applications of the methods described herein, the relativelevels of the various polynucleotides in a sample can be determined andcompared to a reference sequence (i.e., normalized). The term “referencesequence” refers to a nucleic acid sequence serving as a target ofamplification in a sample that provides a control for the assay. Thereference may be internal (or endogenous) to the sample source, or itmay be an externally added (or exogenous) to the sample. A referencesequence is typically amplified during the multiplex amplification. Forexample, when performing gene expression analysis, at least oneamplification target in a multiplex set that is endogenous to the samplecan be selected as a reference sequence. This reference can be a targetthat has been independently shown to exhibit a fairly constantexpression level (for example, a “housekeeping” gene). Examples of suchhousekeeping genes include GAPDH, β-actin, 18SRNA and cyclophilin. Asindicated below, the Ct value from an endogenous reference sequence canprovide a control for converting Ct values of other target sequencesinto relative expression levels. Optionally, a plurality of controltargets/reference sequences that have relatively constant expressionlevels may be included in the multiplexed amplification to serve ascontrols for each other. Alternatively, the reference sequence can be anexternal reference sequence. For example, an external reference sequencemay be a defined quantity of either RNA, added to the sample prior toreverse transcription, or DNA (e.g., cDNA), added prior to the multiplexamplification.

In an example of the use of a reference sequence, a multiplex polymerasechain reaction amplification is carried out to amplify a sample, such asan RNA or a cDNA sample, using a plurality of primer sets for amplifyingtarget sequences, and including a primer set for amplifying a referencesequence. In real-time single-plex amplifications, a Ct^(target) valueis obtained for each target sequence and a Ct^(ref) value is obtainedfor the reference sequence. In order to normalize the Ct^(target)values, the Ct^(ref) value is subtracted from each Ct^(target) value toyield a ΔCt^(target) value for each target sequence. In certainembodiments, the Ct^(pool) value (as described above) can be used as thereference value. For example, the Ct^(pool) value can be obtained andsubtracted from each Ct^(target) value (i.e., the Ct^(pool) value isused in place of the Ct^(ref) value) to yield a ΔCt^(target) value.

Certain applications of the present methods concern analyzing samplesobtained from a cell, cell line, tissue or organism that has undergone atreatment. For example, up-regulation or down-regulation of certaingenes can be analyzed. The term “treatment” refers to the process ofsubjecting one or more cells, cell lines, tissues, or organisms to acondition, substance, or agent (or combination thereof) that may causethe cell, cell line, tissue or organism to alter its gene expressionprofile. A treatment may include a range of chemical concentrations andexposure times, and replicate samples may be generated. The term“untreated control” refers to a sample obtained from a cell, cell line,tissue or organism that has not been exposed to a treatment. mRNA (orcDNA) from an untreated control can be amplified in a multiplexamplification in the same manner as a sample from a treated cell, cellline, tissue or organism. Cycle threshold values obtained from both atreated sample and from an untreated control can be normalized, asdescribed above. For example, a cycle threshold value for the untreatedcontrol (“Ct^(untreated)”) can be obtained for each target sequence inthe untreated control sample and a Ct^(ref) obtained for each referencesequence, as described above. The Ct^(ref) is subtracted from eachCt^(untreated) value to obtain ΔCt^(untreated) values. Similarly, acycle threshold value (Ct^(treated)) for the mRNA (or cDNA) from asample from a treated cell, cell line, tissue or organism can beobtained and normalized to obtain ΔCt^(treated) values. It can be shownthat the amount of target sequence from a treated sample normalized toan endogenous reference and relative to a normalized untreated controlis given by:2^(−ΔΔCt)wherein ΔΔCt=ΔCt^(treated)−ΔCt^(untreated). The ΔΔCt values can be usedin the analysis of the effect of a treatment on the expressed levels oftarget sequences.

In practice, in carrying out any of the multiplex or single-plexamplifications described herein, a passive reference containing afluorescent dye (e.g. ROX) can be included, if desired, to normalize fornon-PCR related fluctuations in the fluorescent signal.

Also disclosed herein is a method for characterizing a plurality ofamplification primer sets that are suitable for amplifying a pluralityof different target sequences of interest. In one embodiment, primersets are pooled, and used in a multiplex amplification in the presenceof a reagent suitable for monitoring the amplification as a function oftime. Examples of such a reagent include oligonucleotide probes. Otherexamples include dye molecules (e.g. intercalating dyes and minor groovebinding dyes).

Also provided herein are reagents and kits suitable for carrying out themultiplex amplification and various two-step reactions and/or assaysdescribed herein. Such reagents and kits may be modeled after reagentsand kits suitable for carrying out conventional PCR and RT-PCRamplification reactions, with the exception that instead of a single setof amplification primers, the reagents and/or kits include a pluralityof amplification primers packaged in a single container, wherein thesingle container may additionally contain one or more oligonucleotideprobes, as described herein. Examples of specific reagents include, butare not limited, to the reagents present in Assays-by-Design™,Pre-Developed Assay Reagents (PDAR) for gene expression, PDAR forallelic discrimination and Assays-On-Demand®, which are commerciallyavailable at Applied Biosystems (an Applera Corporation business). Thekits may optionally include reagents packaged for downstream orsubsequent analysis of the multiplex amplification product. In oneembodiment, the kit includes a container comprising a plurality ofamplification primer pairs or sets, each of which is suitable foramplifying a different sequence of interest, and a plurality of reactionvessels, each of which includes a single set of amplification primerssuitable for amplifying a sequence of interest. The primers included inthe individual reaction vessels can, independently of one another, bethe same or different as a set of primers comprising the plurality ofmultiplex amplification primers. In a specific embodiment, both thecontainer and plurality of reaction vessels further include5′-exonuclease probes such that the kit is, suitable for carrying outthe multistep assay illustrated in FIG. 2. In one embodiment, theplurality of reaction vessels is a multiwell plate.

The invention having been described, various features and advantages ofthe invention are illustrated in the following examples, which areintended to be illustrative and non-limiting.

7. EXAMPLES 7.1 Example 1: Multiplex Amplification Performance Increaseswith Increasing Concentration of DNA Polymerase

To determine the optimal amount of DNA polymerase for performingmultiplex amplifications, 95-plex amplifications were carried out as afunction of DNA polymerase concentration. The amplification primer mixfor the 95-plex amplification was prepared by pooling 10 μL from each of95 different randomly selected 20× Assays-on-Demand™ Gene ExpressionProducts (Applied Biosystems, an Applera Corporation business, CatalogNos. Hs00170531_m1; Hs00176369_m1; Hs00176332_m1; Hs00170586_m1;Hs00173565_m1; Hs00176247_m1; Hs00170192_m1; Hs00177127_m1;Hs00176908_m1; Hs00170380_m1; Hs00173925_m1; Hs00170681_m1;Hs00176394_m1; Hs00170633_m1; Hs00173872_m1; Hs00174690_m1;Hs00170288_m1; Hs00173798_m1; Hs00170423_m1; Hs00174927_m1;Hs00174805_m1; Hs00175976_m1; Hs00176222_m1; Hs00173678_m1;Hs00170261_m1; Hs00173592_m1; Hs00174781_m1; Hs00177401_m1;Hs00173854_m1; Hs00173936_m1; Hs00170248_m1; Hs00173564_m1;Hs00174717_m1; Hs00170407_m1; Hs00174575_m1; Hs00174796_m1;Hs00176315_m1; Hs00170969_m1; Hs00153126_m1; Hs00174765_m1;Hs00153510_m1; Hs00173606_m1; Hs00176075_m1; Hs00170236_m1;Hs00170712_m1; Hs00176239_m1; Hs00176121_m1; Hs00171022_m1;Hs00170174_m1; Hs00173506_m1; Hs00174910_m1; Hs00170210_m1;Hs00174789_m1; Hs00174774_m1; Hs00173773_m1; Hs00174937_m1;Hs00173681_m1; Hs00170903_m1; Hs00176268_m1; Hs00176148_m1;Hs00176865_m1; Hs00174599_m1; Hs00170308_m1; Hs00170823_m1;Hs00176077_m1; Hs00173899_m1; Hs00174860_m1; Hs00173717_m1;Hs00175940_m1; Hs00170684_m1; Hs00173526_m1; Hs00170299_m1;Hs00170991_m1; Hs00176385_m1; Hs00175935_m1; Hs00170403_m1;Hs00173855_m1; Hs00170899_m1; Hs00176202_m1; Hs00170349_m1;Hs00177051_m1; Hs00170472_m1; Hs00173634_m1; Hs00175948_m1;Hs00177552_m1; Hs00175997_m1; Hs00174752_m1; Hs00174674_m1;Hs00176505_m1; Hs00176209_m1; Hs00175999_m1; Hs00176998_m1;Hs00176747_m1; Hs00170433_m1; and Hs00174604 ml. Each 20×Assays-on-Demand™ Gene Expression Product contained two unlabeledamplification primers (18 μM each primer) and one FAM-labeled TaqMan®MGB probe (5 μM). 95-Plex amplifications were carried out with thisamplification primer mix using DNA polymerase concentrations rangingfrom 1 Unit per 20 μL reaction volume (1 U/20 μL) to 17 U/20 μL. For the95-plex amplification carried out with 1 U/20 μL DNA polymerase, 5 μLpooled primer mix, 10 μL 2× TaqMan® Universal PCR Master Mix (“2× MasterMix”; Applied Biosystems, an Applera Corporation business, Cat.#4304437) and 5 μL template cDNA (from a cDNA library; 100 ng totalcDNA) were added to a reaction tube. 2× Master Mix comprises AmpliTaqGold® DNA polymerase (0.1 U/μL), AmpErase® UNG, dNTPs with dUTP, apassive reference and optimized buffer components. 95-Plexamplifications carried out at higher DNA polymerase concentrations wereprepared by spiking the reaction with the appropriate amount of AmpliTaqGold® (5 U/μl; Applied Biosystems Catalog No. N808024). All 95-plexreactions were initially heated (10 min at 95° C.) followed by a totalof 10 cycles (15 sec melt at 95° C.; 1 min anneal/extend at 60° C.) onan ABI Prism® 7700 instrument (Applied Biosystems, an AppleraCorporation business).

The product of each 95-plex amplification was diluted to 200 μl withwater (10-fold) and divided for 95 individual single-plex real-timeamplification reactions. Each single-plex amplification used asprimers/probes one of the 20× Assays-on-Demand™ Gene Expression Productsdescribed above, with a different set of primers per reaction. Thefollowing volumes of reagents were used for the single-plex real-timeamplifications: 2 μL diluted 95-plex amplification product, 1 μL 20×Assays-on-Demand™ Gene Expression Product, 10 μL 2× Master Mix and waterto yield a 20 μL reaction volume. All single-plex amplifications werecarried out for a total of 40 cycles (using the same cycling conditionsas described above) on an ABI Prism® 7700 or 7900 instrument (AppliedBiosystems, an Applera Corporation business). The accumulation ofamplicon was monitored in real time. These amplifications are the “assayamplifications.”

95 corresponding single-plex control amplifications were carried out ina similar manner with template cDNA that had not been subjected tomultiplex preamplification. For each concentration of DNA polymerase,the cycle threshold values (Ct values) of the 95 assay amplificationswere obtained and averaged, yielding an average assay Ct value(Ct^(assay)) for each DNA polymerase concentration. The Ct values forthe 95 control amplifications were also averaged, yielding an averagecontrol Ct value (Ct^(control)). Differences between the averageCt^(assay) values and average Ct^(control) were obtained (ΔCt values)for each DNA polymerase concentration and plotted (FIG. 3). In thisexperiment, multiplex amplifications that perform the same assingle-plex amplifications (with respect to the amount of ampliconproduced) for a particular target sequence yield a ΔCt value of 10. Thecloser the ΔCt to a value of 10, the better the performance of the10-cycle multiplex amplification.

As is evident from FIG. 3, the performance of the 95-plex amplificationincreased with increasing DNA polymerase concentration over a range of1-5 U/20 μL reaction volume, at which concentration the performanceplateaued prior to decreasing slightly. From this experiment, it wasdetermined that the optimal spiked DNA polymerase concentration forcarrying out multiplex amplifications using the reaction conditionsdescribed above is in the range of 4-6 Units per every 20 μL reactionvolume. The decrease in performance observed at higher levels of spikedDNA polymerase is believed to have been caused by exceedingly highconcentrations of components of the enzyme storage buffer, e.g.glycerol, in the multiplex amplification reaction.

7.2 Example 2: Multiplex Amplifications are Efficient at Extremely LowPrimer Concentrations, which do not Require Optimization

Two of myriad advantages of multiplex amplifications is the ability toefficiently amplify in a single reaction multiple sequences usingextremely low primer concentrations and without having to optimizeindividually the concentrations of the primers. To demonstrate thesepoints, 100 ng cDNA was multiplex amplified for 0 cycles or 10 cycles ina 95-plex amplification as described in Example 1, using 6 U/20 μL DNApolymerase. Each multiplex amplification was divided and 95 individualsingle-plex reactions were performed as described in Example 1. The ΔCtvalue (Ct^(0 cycles)−Ct^(10 cycles)) for each single-plex reaction wasobtained and plotted on a bar graph for visual comparison (FIG. 4). Asfor Example 1, the optimal ΔCt for a particular reaction is 10. As canbe seen from FIG. 4, 90 out of 95 of the assays performed well in therandomly-selected multiplex amplification.

Significantly, none of the primer concentrations were optimized for the95-plex amplification step. The commercially available 20×Assays-On-Demand reagents were merely pooled together without furthermanipulation. Moreover, the primer concentrations of the multiplexamplification were extremely low, being present at only 45 nM eachprimer. In contrast, the primer concentrations used for the single-plexamplifications were 900 nM each primer.

7.3 Example 3: Multiplex Amplifications can be Carried Out in thePresence of Oligonucleotide Probes

Another significant advantage of multiplex amplifications is the abilityto carry out the reaction in the presence of oligonucleotide probeswithout significant interference during either the multiplexamplification or downstream amplifications carried out on the multiplexamplification product. This former advantage is apparent from Example 2,supra. In Example 2, efficient amplification was achieved in themultiplex amplification step, which by virtue of utilizingAssays-On-Demand™ reagents to create the multiplex primer pool, includedTaqMan® MGB oligonucleotide probes in the reaction.

To demonstrate the latter advantage, a single-plex RNase P assay (DNAspecific) was run with 1 ng of genomic DNA in the presence or absence ofa 5× concentration of the 95-plex primer/probe pool (RNA-cDNA specific)described in Example 1. The 5× concentration of 95-plex primer/probepool was added to determine what effect it would have on the single-plexRNase assay. The average Ct values of the two reactions are illustratedin FIG. 5. As evident from FIG. 5, the presence of the 95-plexprimer/probe pool did not affect the Ct value of the RNase P amplicon,demonstrating that single-plex RNase P assays can be carried out withthe product of a multiplex amplification reaction without having tofirst remove the multiplex primers and/or probes. The presence of themultiplex primers and/or probes does not deleteriously affect theperformance of the single-plex RNase P assay.

7.4 Example 4: Multiplex Amplification Permits the Downstream Analysisof Very Small Amounts of Sample

To demonstrate that multiplex amplifications permit downstream analysisof quantities of sample that would otherwise be too small for thedesired type and/or number of analyses, multiplex amplifications werecarried out with varying concentrations of sample cDNA ranging from 100ng to 100 pg (approximately equivalent to a sample size of 5 cells).Each concentration of cDNA was subjected to 95-plex amplificationfollowed by 95 individual real-time amplification analyses as describedin Example 1. The average Ct values of the 95-plex amplifications as afunction of sample cDNA concentration are provided in FIG. 6. Asillustrated in FIG. 6, there is a linear relationship between the samplecDNA concentration and average Ct value, demonstrating that a largepercentage (approx. 97%) of the target sequences amplified efficiently,even though they were amplified simultaneously in a multiplexed fashion.The level of sensitivity achieved demonstrates that samples from as fewas 1-2 cells can be analyzed by real-time PCR following multiplexamplification. Moreover, the multiplex amplification yields a quantityof amplified sample sufficient for numerous downstream real-time PCRassays.

7.5 Example 5: Multiplex Amplification at Increased Multiplexity

To demonstrate that multiplex amplifications can be carried out at veryhigh levels of complexity, 186-plex, 369-plex, 738-plex and 1013-plexamplifications were carried out in four individual multiplexamplification reactions. The amplification primer mix for each of theamplifications was prepared by pooling equal volumes of 186, 369, 738 or1013 different randomly selected 20× Assays-on-Demand™ Gene ExpressionProducts into four separate microcentrifuge tubes, respectively. Foreach of the four tubes, the pooled solution was dried using a SpeedVac®concentrator (Thermo Savant, Holbrook, N.Y.). The residue wasre-suspended in deionized water such that the multiplexed amplificationprimers were at a 4× stock concentration (180 nM each primer) relativeto the 1× working amplification primer concentration of 45 nM. For the1013-plex pooled mixture, the combined primers were present in there-suspension at a concentration of 45.6 μM, and the FAM-labeled TaqManMGB probes were present at 10.1 μM. For the 186-plex amplification, 92primer sets from each of two plates (designated IAP and IAO) were pooledalong with equal volumes of primer sets for two reference genes,glyceraldehyde phosphate dehydrogenase (GAPDH) and cyclophilin. Insetting up the experiment described in this Example, for convenience inliquid transfers, each of the above randomly selected 20×Assays-on-Demand Gene Expression Products was distributed into a seriesof 96-well plates (designated alphabetically plates IAA through IAO).Each 20× Assays-on-Demand Gene Expression Product contained twounlabeled amplification primers (18 μM each primer) and one FAM-labeledTaqMan® MGB probe (5 μM).

Each of the amplifications (from the 186-, 369-, 738- or 1013-plexpooled primer mixtures) were carried out in a final volume of 50 μL,with the constituents being 12.5 μL of 4× pooled and re-suspended primermix, 25 μL 2× TaqMan® Universal PCR Master Mix (“2× Master Mix”; Cat.#4324016 containing no UNG enzyme), 10 μL template cDNA (from a cDNAlibrary; 25 ng total cDNA) and 2.5 μL AmpliTaq Gold® DNA polymerase (5U/μL). The 2× Master Mix included AmpliTaq Gold® DNA polymerase (0.1U/μL), dNTPs, a passive reference and optimized buffer components. Eachof the four reactions were carried out for a total of 10 cycles (15 sec.melt at 95° C.; 4 min. anneal/extend at 60° C.) on an ABI Prism® 7700instrument.

The product of each amplification was diluted with water and aliquotedfor single-plex analysis. In the case of the 186- and 369-plexreactions, the product was diluted 1:5 prior to setting up thesingle-plex assays. For the 738- and 1013-plex amplifications, theproduct was diluted 1:10 prior to setting up the single-plex assays.Each of these single-plex amplifications used as primers/probes one ofthe 20× Assays-on-Demand™ Gene Expression Products used in the multiplexamplification described above. and were distributed into a series of96-well plates for liquid transfer convenience (designatedalphabetically plates IAA through IAO). A different set of primer/probeswas used in each single-plex reaction. The following volumes of reagentswere used for the single-plex (“assay”) amplifications: 2.5 μL diluted186-, 369-, 738- or 1013-plex amplification product, 0.5 μL 20×Assays-on-Demand Gene Expression Product, 5 μL 2× Master Mix and waterto yield a 10 μL reaction volume. All assay amplifications were carriedout for a total of 40 cycles (15 sec. melt at 95° C.; 1 min.anneal/extend at 60° C.) on an ABI Prism® 7900 instrument. Theaccumulation of amplicon was monitored in real time.

Corresponding multi-plex control, 186-plex, 369-plex, 738-plex and1013-plex control amplifications were prepared as described above, butthese control multiplex amplifications were not subjected to thermalcycling (i.e., the reactions were not subjected to multiplexamplification). These “mock” amplification mixtures were diluted andassayed in single-plex “control” amplifications as described above. Foreach assay amplification, the Ct values of the targets from plates IAOand IAP were obtained and averaged, yielding an average assay Ct value.Average ΔCt values were calculated as described in Example 1. The ΔCtvalues are sorted by ascending ΔCt value in FIG. 7.

The following table summarizes the results from the various assayamplifications:

Multiplex Reaction Avg. ΔCt value Std. Dev. Median 186-plex 9.86 0.479.84 368-plex 10.01 0.82 10.10 738-plex 9.94 0.43 9.97 1013-plex  9.980.67 10.10The results demonstrate that there was no degradation in performance incarrying out multiplex amplifications at very high levels of complexity.

7.6 Example 6: Multiplex PCR Amplifications can be Carried Out in thePresence of Uracil N-Glycosylase (UNG)

A method for preventing “carry over” contamination in PCR includes theuse of dUTP in place of dTTP in the PCR mixture, followed by treatmentof all subsequent PCR mixtures with uracil N-glycosylase (UNG) (U.S.Pat. No. 5,035,996). The experiment in this Example was performed inorder to evaluate the effect of the presence of UNG on the efficiency ofmultiplex amplifications.

A first 186-plex amplification (UNG(−)) was carried out as described inExample 5, but using TaqMan® Universal Master Mix, No AmpErase® UNG(Cat. #4324018), instead of Universal Master Mix (Cat. #4304437). Themultiplex amplification was extended for 14 cycles, instead of 10cycles, as in Example 5. The samples were chilled on ice after theamplification, and then subjected to single-plex PCR as described inExample 5.

Another 186-plex amplification (UNG(+)) was carried out as described inExample 5, but using Universal Master Mix (Cat. #4304437) (with UNG),except that the 186-plex amplification was extended through 14 cycles,and the samples were chilled on ice for 4 hours. The samples weresubjected to single-plex PCR as described in Example 5.

For each of these two protocols, corresponding control multiplexamplifications were set up, but these control reactions were notsubjected to thermal cycling. The ΔCt value for the single-plexamplification from the “UNG(−)” protocol and from the “UNG(+)” protocolwere obtained and plotted in a bar graph (FIG. 8). The following tablesummarizes the results of the two protocols:

Avg. ΔCt Std. Dev. Median UNG(−) 14.13 0.62 14.08 UNG(+) 13.94 0.8113.96

In this Example, the effect of the presence of UNG in a multiplexamplification, carried out in a procedure similar to that described inExample 5, was evaluated. The presence of UNG in the multiplexamplification had essentially no affect on the efficiency ofamplification in the single-plex amplification step as compared to theUNG(−) sample.

7.7 Example 7: Effect of Increased Cycles of Multiplex Amplification

In Example 5, the multiplex amplification was carried out for 10 cycles.In the present Example, multiplex amplifications were carried out for10, 12 and 14 cycles. Higher cycle numbers can increase theconcentration of the amplification product, which allows a greaternumber of downstream assays, such as a greater number of single-plexamplifications, to be performed.

Three 186-plex amplifications were carried out as described in Example5, using Universal Master Mix (Cat. #4324018) (with no AmpErase® UNG),and the multiplex amplification was extended through 10, 12 or 14amplification cycles. ΔCt values for each the three protocols is shownin FIG. 9 and summarized in the following table.

Cycles Avg. ΔCt value Std. Dev. Median 10 9.99 0.59 9.92 12 11.80 0.5611.78 14 14.13 0.60 14.08

The average ΔCt value increased as the number of thermal cyclesincreased. The standard deviations were essentially unchanged. Theseresults indicate that there is no decrease in performance in going from10 to 14 cycles. If amplifications are 100% efficient, then the ΔCtbetween amplified and “mock” reactions will be 10 with 10 cycles ofamplification, 12 with 12 cycles, and 14 with 14 cycles. In thisExample, the average ΔCt values approximated 10 (9.99), 12 (11.8), and14 (14.13), respectively.

7.8 Example 8: Quantification of mRNA in Human Plasma by RT-PCRMultiplex Amplification

The detection of circulating RNA in plasma can allow for early detectionof disease states such as cancer, coronary and autoimmune dysfunctionsand can also be used to monitor the success of drug treatment regimes byfollowing gene expression.

This Example demonstrates generation of cDNA from a sample of mRNAfollowed by multiplex PCR amplification of the cDNA in the presence of aplurality of selected PCR primers within a single reaction mixture, withsubsequent single-plex real-time PCR in the presence of each of theselected PCR primers.

Whole blood was obtained from a healthy donor and spun at 2000×g for 20min. The cell-free supernatant was decanted and filtered through a 0.2μm filter. The recovery (volume approximately 10 ml) was about 50% ofthe whole blood volume. RNA was extracted and subjected to ethanolprecipitation. The RNA pellet was re-suspended in 200 μl of TE buffer.

Equal volumes (10 μl each) of 108 selected 20× Assays-on-Demand GeneExpression Products were pooled and dried down using a SpeedVac®concentrator (Thermo Savant, Holbrook, N.Y.). The residue wasre-suspended in deionized water to yield a primer concentration of 180nM for each primer. The 108 Assay-on-Demand primers corresponded tosolid tissue and leukocyte specific amplicons, examples of whichincluded: pinin, hexokinase-1, VEGFβ, PRKCB1, LGALS3BP, cyclophilin A,GAS2L1, DDX1, TERT, BMPR2, LANCL1, and CCL5.

The reverse transcriptase (RT) and multiplex amplification incubationcontained the following: 125 μl of 2× Universal Master Mix for one-stepRT-PCR (TaqMan® One Step Master Mix Reagents Kit, No AmpErase® UNG, Cat.#4309169); 12.5 μl AmpliTaq Gold (5 U/μl=5 Units extra per 20 μlmultiplex amplification volume); 6.25 μl of reverse transcriptase/RNaseinhibitor; 106.25 μl plasma RNA (240 ng); and 62.5 μl of the pooled andre-suspended 108 Assay-on-Demand Products. The final volume was 250 μl(240 ng plasma RNA), and 50 μl was aliquoted into separate wells of a96-well plate.

The reverse transcription reaction was carried out for 30 min at 48° C.,followed by denaturation for 10 min at 95° C. The multiplexamplification was carried out for a total of 14 cycles (each cycle: 15sec at 95° C.; 4 min. anneal/extend at 60° C.). These reactions werecarried out on an ABI Prism® 7700 instrument.

In “mock” multiplex amplifications which included the reversetranscription reaction, but in which the 14 cycles of PCR multiplexamplification were omitted, no signal due to specific amplicons could bedetected. The following table summarizes the results for selectedtargets:

Target Ct 18S 20.36 (0.03) Pinin 32.83 (0.54) Hexokinase-1 32.10 (0.62)VEGFβ 34.20 (0.38)

The results indicate that DNA obtained from reverse transcription ofhuman plasma RNA, and subjected to multiplex amplification in thepresence of a plurality of primer pairs, can subsequently be single-plexamplified using the same primer pairs without optimization.

Unless mentioned otherwise the techniques employed or contemplatedherein are standard methodologies well known to one of ordinary skill inthe art. The materials, methods and examples are illustrative only andnot limiting.

While the foregoing has presented specific embodiments of the presentinvention, it is to be understood that these embodiments have beenpresented by way of example only. It is expected that others willperceive and practice variations which, though differing from theforegoing do not depart form the spirit and scope of the invention asdescribed and claimed herein. All patent applications, patents, andliterature references cited in this specification are herebyincorporated by reference in their entirety.

What is claimed is:
 1. A kit for carrying out a two-step amplificationsreaction comprising: (a) a first container or reaction vessel comprisinga plurality of amplification primer sets suitable for carrying out amultiplex amplification reaction to produce a plurality of differentamplicons and one or more single-stranded oligonucleotide probescomplementary to all or a part of one or more of the plurality ofdifferent amplicons; and (b) a second container or reaction vesselcomprising a single set of amplification primers suitable for carryingout a singleplex amplification reaction to produce a single amplicon,and a single-stranded oligonucleotide probe complementary to all or apart of the single amplicon.
 2. The kit of claim 1, wherein the singleset of amplification primers in the second container or reaction vesselis the same as or nested to one of the plurality of amplification primersets in the first container or reaction vessel.
 3. The kit of claim 1,wherein the plurality of amplification primer sets comprises at least100 different amplification primer sets.
 4. The kit of claim 1, whereinthe kit further comprises a third container or reaction vesselcomprising a DNA polymerase.
 5. The kit of claim 1, wherein the kitfurther comprises a third container or reaction vessel comprising areverse transcriptase.
 6. The kit of claim 1, wherein at least 60% ofthe plurality of amplification primer sets in the first container orreaction vessel are at approximately an equimolar concentration relativeto one another.
 7. The kit of claim 6, wherein all of the plurality ofamplification primer sets in the first container or reaction vessel areat approximately an equimolar concentration relative to one another. 8.The kit of claim 1, wherein the concentration of each of the pluralityof amplification primer sets is at a concentration less than 100 nM foreach primer.
 9. The kit of claim 1, wherein the concentration of each ofthe plurality of amplification primer sets is at a concentration ofabout 30 to 45 nM for each primer.
 10. The kit of claim 1, wherein theone or more single-stranded oligonucleotide probes in the firstcontainer or reaction vessel is a TaqMan probe.
 11. The kit of claim 1,wherein the single-stranded oligonucleotide probe in the secondcontainer or reaction vessel is detectably labeled.
 12. The kit of claim1, wherein the single-stranded oligonucleotide probe in the secondcontainer or reaction vessel is a TaqMan probe.
 13. The kit of claim 1,wherein at least one step of the two-step amplification reaction is areverse transcription polymerase chain reaction (RT-PCR).
 14. The kit ofclaim 1, wherein the first container or reaction vessel furthercomprises a DNA polymerase and/or a reverse transcriptase.
 15. The kitof claim 1, wherein the second container or reaction vessel furthercomprises a DNA polymerase and/or a reverse transcriptase.
 16. The kitof claim 4, wherein the DNA polymerase is at a concentration of about4-6 U/20 μL reaction volume.
 17. The kit of claim 8, wherein theconcentration of the single set of amplification primers in the secondcontainer or reaction vessel is at a concentration of about 300-900 nMfor each primer.
 18. The kit of claim 2, wherein the single set ofamplification primers in the second container or reaction vessel and oneof the plurality of amplification primer sets in the first container orreaction vessel are the same.
 19. The kit of claim 1, wherein theplurality of amplification primer sets in the first container orreaction vessel and the single set of amplification primers in thesecond container or reaction vessel all have a Tm that is within 10degrees of one another.
 20. The kit of claim 1, wherein the one or moresingle-stranded oligonucleotide probes in the first container orreaction vessel is as the same as the single-stranded oligonucleotideprobe in the second container or reaction vessel.